Chemical toxicity testing method based on self-research and development pollutant exposure system

Abstract

The invention belongs to the technical field of chemical biotoxicity testing, and particularly relates to a chemical toxicity testing method based on a self-developed pollutant exposure system. According to the testing method, a self-developed pollutant exposure system is used for simulating the toxic effect of chemicals in a natural water body, zebra fish is adopted as a model organism, a time-concentration linear function of to-be-tested chemicals in a contaminated area is established, behavior analysis is carried out through observation of abnormal behaviors of the zebra fish or a coupled camera tracing technology, and the method is used for determining the toxic effect of the to-be-tested chemicals in the contaminated area. And calculating the minimum effect concentration of the to-be-tested chemical. The test method is almost a non-destructive experiment, the experiment time is short, most zebrafish participating in the experiment can be recovered to a normal state through proper recovery, the time cost is greatly saved, the efficiency is high, and the death rate of tested organisms is low.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical biological toxicity testing, and in particular relates to a chemical toxicity testing method based on a self-developed pollutant exposure system. Background Art

[0002] At present, the aquatic toxicity test data of chemicals are mainly reflected in the median lethal concentration (LC50). 50 ) and the median effective concentration (EC 50 ), these data often require a long testing time (such as 24h, 48h, 96h), and have irreversible damage to model organisms (such as death); in addition, there are molecular toxicity data such as carcinogenicity, mutagenicity, neurotoxicity, reproductive toxicity, and endocrine toxicity. The testing of these toxicity data requires strong professional knowledge and skills, and the instruments and conditions required for the testing are relatively expensive. Some experiments require exposure of animals and extraction of target organs and tissues, which also cause irreversible damage to model organisms.

[0003] Chemicals enter environmental water bodies through various pathways, and their concentrations are not constant. The dilution effect will reduce the concentration of chemicals entering the water body, which makes it difficult to set control concentrations for discharge and leakage. To obtain such data, it is necessary to conduct steady-state exposure experiments with multiple concentration gradients, long periods of time, and high lethality, which greatly consumes manpower and material resources. Therefore, it is necessary to develop an experimental method that can accept and dilute chemicals in natural water bodies and conduct gradual and cumulative exposure of organisms in the water to guide chemical management.

[0004] Animal behavior experiments are an experimental method that compares the differences in behavior between normal and abnormal states. They are widely used in toxicology research. Because the behavior needs to be recorded, the number of animals used and the number of animal deaths are much lower than LC 50 In the test experiment, some animals can return to normal after an appropriate recovery period. At the same time, fish use a variety of sensory organs to sensitively perceive changes in water flow and water quality, and make a variety of behavioral responses to complete the foraging, risk avoidance, and courtship behaviors necessary for survival and reproduction. Behavioral changes are more sensitive than biochemical biomarkers. Moreover, behavioral abnormalities may also be caused by motor nerve abnormalities, which can be caused by neurotoxicity or endocrine homeostasis imbalance, and the time is much earlier than death, which has an early warning function.

[0005] The current aquatic biological toxicity testing technology is relatively simple, the molecular biology toxicity testing technology requires a high level of professionalism, and the biological damage and mortality rate of the testing method are also relatively high. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for testing the toxicity of chemicals based on a self-developed pollutant exposure system. This method借助自研发的污染物暴露系统,用于模拟化学品在天然水体中的毒性作用,采用斑马鱼作为模式生物,通过对斑马鱼异常行为的观测或耦合摄像示踪技术进行行为分析,得到产生持续时间较长的异常行为而不致斑马鱼死亡的浓度,具有方法简单、实验时间短、生物死亡率低、受试生物可恢复等优势。采用的技术方案为:

[0007] A method for testing the toxicity of chemicals based on a self-developed pollutant exposure system, which uses a self-developed pollutant exposure system. The system includes a cylinder with an open top. Inside the cylinder, a buffer zone, a contamination zone, and an effluent zone are sequentially arranged. The buffer zone and the contamination zone, as well as the contamination zone and the effluent zone, are separated by perforated partition plates. Zebrafish are used as model organisms and placed in the exposure system. The testing method specifically includes the following steps:

[0008] (1) Mother liquor preparation. Query the LC 50 value of the chemical to be tested, and adjust it according to the LC 50 value of the chemical to be tested to configure the initial concentration of the mother liquor. If the behavioral toxicity of the chemical to zebrafish is not obvious, the concentration can be further increased according to the results of the preliminary experiment;

[0009] (2) Establish a time-concentration linear function of the chemical to be tested in the contamination zone, which is:

[0010] When t ≤ 40 minutes, C = 0.60871t + 0.36129;

[0011] When 40 minutes < t ≤ 600 minutes, C = 0.04706t + 24.10669;

[0012] where C is the concentration percentage of the chemical relative to the mother liquor, in %, and t is the inlet time of the mother liquor;

[0013] (3) Conduct a significant behavior test in the exposure system, and calculate the concentration at which the chemical to be tested causes significant changes in the behavior of zebrafish, that is, the lowest effect concentration;

[0014] (4) Conduct behavioral analysis on the zebrafish in the exposure system through a coupled camera tracing technique to obtain the lowest effect concentration of the chemical to be tested;

[0015] Among them, steps (3) and (4) are selective analysis steps.

[0016] Preferably, the initial concentration of the chemical mother liquor is adjusted according to the LC 50 value of the chemical to be tested, and the adjustment range is 0.5LC 50~2LC 50 .

[0017] Preferably, the partition spacing of the self-developed pollutant exposure system is not less than 20 cm, holes are punched on the partitions, and an internal circulation system is arranged in the buffer zone, the contaminated zone and the water outlet zone; a water inlet pipe is arranged in the buffer zone, and a water outlet pipe is arranged in the water outlet zone, and both the water inlet pipe and the water outlet pipe are connected to a peristaltic pump to provide power, and the inlet and outlet water flow rates are set at 0.1 to 10 L / h.

[0018] As a further preference, the internal circulation system includes a submersible pump, a circulating water pipe and an aeration component, and the aeration component includes an aeration pipe and an aeration regulating valve; the circulation flow rate of the submersible pump is 500-1500L / h, the submersible pump is placed in the middle of the cylinder wall in the water outlet area, and maintains a distance of 1-3mm from the cylinder wall, and the pump body and the cylinder body are filled with biochemical cotton to absorb excrement; the circulating water pipe is connected to the water outlet of the submersible pump, and the other end is connected to the buffer zone, the water outlet end of the circulating water pipe is vertically downward, the water outlet is located in the middle of the buffer zone, and a hole is opened 0.5-1cm above the water outlet of the circulating water pipe, the aeration pipe and the aeration regulating valve are inserted, and the aeration regulating valve is placed in the air, and a tubular hard plastic thin tube with an inclined cut is used at the interface, and the shorter side faces the water outlet of the circulating water pipe to ensure that the water flow does not enter the aeration pipe.

[0019] As a further preference, the partitions include an upper perforated partition, a lower perforated partition, and a fully perforated partition. The partitions are made of transparent acrylic sheets, and a 1 to 3 mm high manure trough is left at the bottom.

[0020] As a further preference, the aperture of the holes on the partition is 1 to 3 mm, and the distance between the holes is set to 1 to 2 times the aperture diameter.

[0021] Preferably, the time-concentration linear function of the chemical to be tested is obtained by fitting a scatter plot of pollutant concentration and time measured at different time points in each region, and the fitting method is apparent fitting.

[0022] Preferably, the method of the significant behavior test is:

[0023] Pure water is pre-filled in the cylinder, preheated to 27°C using a heating rod, sterilized with an ultraviolet lamp and aerated for more than 1 hour; adult male zebrafish are placed in the contaminated area to adapt to the water quality, and the adaptation stage is at least 1 hour; after the adaptation period, a blank control experiment is conducted for 1 hour, that is, the inlet and outlet peristaltic pumps are turned on, and the inlet peristaltic pump is connected to pure water. The behavior of the zebrafish during the blank control experiment is observed in detail as a standard behavioral control. If it is necessary to observe the toxic target in a targeted manner, a stimulation experiment of the corresponding toxic target should be additionally conducted. For example, to determine the visual damage of chemicals to zebrafish, light stimulation or image stimulation without noise and vibration should be conducted, and the behavioral response of the zebrafish should be observed;

[0024] After the blank control experiment, the water inlet pipe is directly connected to the mother solution of the chemical to be tested to carry out the poisoning experiment. During the experiment, the normal behavior and abnormal behavior changes of the zebrafish are observed. The obvious abnormal postures need to be photographed and recorded, and the time when the abnormal behavior occurs is also recorded;

[0025] If there is no significant abnormal behavior within 9 hours, increase the concentration of the mother solution of the chemical to be tested for a second experiment. The concentration of the mother solution of the chemical to be tested can be appropriately increased, for example, by 20%, 50%, etc. of the initial concentration, until abnormal behavior occurs;

[0026] If significant abnormal behavior occurs within 9 hours, the time of the abnormal behavior is recorded and the minimum effect concentration is calculated based on the time-concentration linear function of the test chemical.

[0027] Preferably, the standard / normal behaviors include sinking, floating, cruising, and hitting the wall, but are not limited to these four behaviors; the abnormal behaviors include sudden high-speed movement, long-term abnormal high-speed / stationary state, and change in swimming posture. The selection of abnormal behaviors should have significant differences to avoid differences in human subjective judgment.

[0028] Preferably, for the test chemicals with lower toxicity, their behavior changes are analyzed by coupling video tracing technology to obtain non-lethal abnormal behavior concentration, that is, the minimum effect concentration.

[0029] Preferably, the behavior analysis method of the coupled video-tracing technique is:

[0030] Install a camera at a suitable height above the center of the cylinder to take pictures of the inside of the cylinder;

[0031] First, shoot a 1-2 hour blank control video as a normal baseline, and then add the chemical mother solution into the water until the zebrafish behavior becomes abnormal or the chemical mother solution has been added to the water for 10 hours;

[0032] The zebrafish were fished out and placed in sterilized pure water to recover. The video was analyzed to quantitatively obtain behavioral intensity data such as the swimming trajectory, swimming speed, distance and time of the zebrafish. By comparing with the baseline, long-term abnormal movement state was selected as an indicator of abnormal behavior, and the minimum effect concentration was calculated based on the starting time of this abnormal movement state.

[0033] Preferably, a high-definition camera with a resolution of at least 1288×964 and a frame rate of 30 frames per second is selected, and the camera can clearly capture the exposure system.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] With the help of a self-developed pollutant exposure system, the present invention can simulate the discharge of chemicals from point sources into larger water bodies, gradually increase the concentration from low to high concentrations in a gradual and cumulative manner, and disperse pollutants with higher efficiency. At the same time, the chemical concentration-time linear function model disclosed in the present invention can support concentration prediction for the entire time period.

[0036] The present invention uses a self-developed pollutant exposure system to perform chemical toxicity testing, which can reflect the neurotoxicity of chemicals to zebrafish or characterize behavioral changes caused by endocrine disorders through the lowest effective concentration of abnormal behavior. The experimental method is almost a non-destructive experiment. Most of the zebrafish participating in the experiment can be restored to normal through appropriate recovery, saving the cost of test animals. Since the exposure mode is gradual and cumulative, a response can be obtained relatively quickly. Zebrafish will show more significant abnormal behavior at concentrations below the half lethal concentration; for those chemicals with lower toxicity, their behavioral changes can be analyzed through coupled video tracing technology to obtain non-lethal abnormal behavior concentrations, greatly saving time costs, being highly efficient and having a low mortality rate of test organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the structure of the self-developed pollutant exposure system used in the present invention (camera omitted);

[0038] Figure 2 A schematic diagram of a partition of a self-developed pollutant exposure system used in the present invention;

[0039] Figure 3 is the functional equation for the change in concentration of the pollutant (chemical to be tested) in the three regions of the exposure system;

[0040] Figure 4 This is a flow chart of the significant behavior test in Example 1;

[0041] Figure 5 This is a flow chart of the behavior analysis of the coupled imaging and tracing technology in Example 2;

[0042] Figure 6 The trajectory diagram of zebrafish at different time periods when ZnSO4 enters the water in Example 2;

[0043] Figure 7 This is a schematic diagram of the movement distance of ZnSO4 water inflow-water inflow time in Example 2;

[0044] Figure 8 This is a schematic diagram of the movement time-water inflow time of K2Cr2O7 water in Example 3;

[0045] Fig. 9 This is a schematic diagram of the recordable exercise time-water inflow time every 5 minutes when K2Cr2O7 is inflowed with water in Example 3;

[0046] Fig.10 Schematic diagram of the number of movements, time, distance and water inflow time of lead acetate in Example 4 DETAILED DESCRIPTION

[0047] The drawings are for illustrative purposes only. To better illustrate the present embodiment, some parts of the drawings are omitted, enlarged or reduced, and do not represent the size of the actual product. It should be understood that the directions or positional relationships indicated by directional words such as "up", "down", "left", "right", "side", "outside", "lateral", "vertical", "horizontal", "top", "bottom", etc. are usually based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present application and simplifying the description. Unless otherwise specified, these directional words do not indicate or imply that the devices or elements referred to must have a specific direction or be constructed and operated in a specific direction, and therefore cannot be understood as limiting the scope of protection of the present application. The directional words "inside and outside" refer to the inside and outside relative to the outline of each component itself.

[0048] For those skilled in the art, some well-known structures and their descriptions in the drawings may be omitted, and therefore, they should not be understood as limitations of the present invention.

[0049] The present invention is further described in detail below in conjunction with embodiments and drawings.

[0050] Embodiment 1:

[0051] like Figure 1 As shown, the present invention uses a self-developed pollutant exposure system, including a cylinder body with an open top, wherein the cylinder body is sequentially provided with three areas, namely, a buffer zone 1, a contaminated zone 2, and a water outlet zone 3. A water inlet partition 11 is provided between the buffer zone 1 and the contaminated zone 2, and a water outlet is provided between the contaminated zone 2 and the water outlet zone 3. The three areas are separated by partitions, and holes are punched on each partition to allow water and pollutants to pass through. The designed aperture is 2 mm. In order to enhance the pollutant diffusion effect, the distance between the holes is set to 1.5 times the aperture. All aquatic organisms smaller than this size can use the device for experiments. Each partition uses a fixed buckle made of acrylic sheet and a special suction cup for breeding (i.e., the first fixed suction cup 8) to fix the position of the partition. The buckle and suction cup should be placed on one side of the buffer zone 1 and the water outlet zone to prevent affecting the normal swimming and shooting of the organisms.

[0052] The buffer zone 1 is provided with a sewage inlet pipe 7, the other end of which is connected to a sewage tank that is filtered and kept at a constant temperature using a heating rod, and the outlet zone 3 is provided with an outlet pipe 13, the other end of which is connected to a wastewater collection system. The sewage inlet pipe 7 and the outlet pipe 13 are both connected to a peristaltic pump to provide power.

[0053] The buffer zone 1, the contaminated zone 2 and the water outlet zone 3 are provided with an internal circulation system.

[0054] The internal circulation system includes a submersible pump 10, a circulating water pipe 9 and an aeration assembly 4; the submersible pump 10 adopts the same submersible pump as the common freshwater fish farming filtration system on the market, and the circulation flow rate is 500-1500L / h, and the flow rate can be set within this range. The submersible pump 10 is placed in the middle of the cylinder wall in the water outlet area 3, and is fixed with an acrylic fixing buckle and a second fixing suction cup. The pump outlet is vertically upward, the water inlet section faces the cylinder wall, and maintains a distance of 3mm from the cylinder wall, and biochemical cotton is filled between the pump body and the cylinder body to absorb excrement.

[0055] The circulating water pipe is connected to the outlet of the submersible pump, and the other end is connected to the buffer 1, that is, the outlet of the circulating water pipe 9 is set in the buffer 1. A hole is opened 1 cm above the outlet of the circulating water pipe 9, and the aeration pipe 5 and the aeration regulating valve 6 are inserted, and the aeration regulating valve 6 is placed in the air. The interface uses a tubular hard plastic thin tube with a bevel cut, and the shorter side faces the outlet of the circulating water pipe 9 to ensure that the water flow does not enter the aeration pipe 5.

[0056] like Figure 2 As shown, as a further preference, the water inlet baffle 11 adopts a fully open-hole baffle, and the water outlet baffle 12 adopts a lower open-hole baffle.

[0057] The openings on each baffle can meet the conditions of water distribution systems and pollutants of different specifications. At the same time, this design can also meet the direct exposure of organisms that adapt to different water depths. For example, benthic mussels can use the lower perforated baffle.

[0058] A high-definition camera with a resolution of 1288×964 and a frame rate of 30 frames per second is selected, and the camera can clearly shoot the exposure system. The camera is installed and fixed using existing technology, and its installation position is located at a suitable height above the exposure system shown, so as to be able to shoot clearly.

[0059] In this embodiment, the design size of the cylinder body is 40×23×25cm, and the cylinder thickness is 5mm; each partition is vertically inserted into the cylinder body, dividing the long side of the cylinder into an 8cm buffer zone, a 22cm contaminated zone and a 9cm outlet zone; the sewage inlet pipe and outlet pipe are 12cm away from the bottom of the cylinder; the sewage inlet pipe is 1cm away from the outlet end of the circulating water pipe, and the aeration hole is 1cm away from the outlet end of the circulating water pipe; the bottom of the submersible pump is 8cm away from the bottom of the cylinder.

[0060] The partition size is 22×24cm, the bottom manure trough is 2mm high, the whole board is perforated with a hole diameter of 2mm and a thickness of 5mm.

[0061] The width of the swimming organisms used in the experiment is not less than 3mm, so the bottom feces trough and the aperture are both less than 3mm; the tank body is 23cm wide and the partition is 22cm wide. In the event of manufacturing errors, the fit is tight and can effectively prevent organisms with strong swimming ability from leaving the contaminated area from both sides of the partition; the partition height is designed to be slightly lower than the tank body height to prevent manufacturing errors from causing it to be higher than the tank body and affecting the subsequent installation of the cover; the cover is optional and its main purpose is to prevent fish from jumping out of the tank body.

[0062] Since fish and shrimps tend to jump out of the water, the water surface needs to be lower than the height of the baffle / tank to prevent them from leaving the contaminated area or jumping out of the device from above. To meet the vertical swimming distance of aquatic organisms, the water surface height is designed to be 20 cm, the size of the contaminated area is 22×23×20 cm, and the volume is 10.12 L.

[0063] Linear fitting is achieved by Origin software, and the fitting method is apparent fitting, thereby obtaining the time-concentration change function equation of the pollutant (chemical to be tested) in the three regions of the exposure system as follows: Figure 3 As shown (x and y represent the values ​​of the horizontal and vertical axes. In the time-concentration change function equation, y is the pollutant concentration, that is, C, and x is the time, that is, t), the minimum effect concentration corresponding to all abnormal behaviors is obtained through the concentration function of the contaminated area (the water body directly exposed to the zebrafish) and the time when the abnormal behavior occurs.

[0064] In this embodiment, a camera is used to record the behavior of zebrafish without analyzing it, and abnormal behavior visible to the naked eye is used as a judgment standard, which has a certain indicative effect on the minimum effective concentration of chemicals with higher toxicity.

[0065] like Figure 4 As shown in the figure, phenol was used as a model chemical. According to the literature, the LC value of phenol on adult zebrafish is 50 The concentration of phenol stock solution was 144.02μM. The pre-experiment was conducted using the phenol stock solution with this concentration as the influent. It was found that it took a long time for the zebrafish to show abnormal behavior, and within the first 20 minutes of influent, the zebrafish did not show significant avoidance behavior. Therefore, we increased the concentration of the phenol stock solution to 2mM and set the influent flow rate to 1L / h.

[0066] The tank was pre-filled with pure water, which was preheated to 27°C using a heating rod, sterilized with an ultraviolet lamp and aerated for 1 hour; adult zebrafish (3 months) were placed in the contaminated area to adapt to the water quality, and the adaptation phase lasted for 1 hour; after the adaptation period, the peristaltic pump was turned on to connect pure water as the inlet water, and a blank control experiment lasting 1 hour was conducted.

[0067] During the adaptation phase, zebrafish showed a stable speed of sticking to the wall, indicating that they had a range of knowledge about the experimental waters. They were almost never completely still for more than 1 minute, and they floated up and down normally, and their swimming posture was normal, which were all considered normal behaviors. Exposure to organic matter may cause zebrafish to become excited and swim quickly, swim vertically, swim sideways, struggle and become anesthetized. Phenol, as a representative of organic matter, may have the above behaviors, so special attention should be paid during observation.

[0068] After 1 hour, the water inlet pipe was directly connected to the phenol mother solution for a poison exposure experiment. In the experiment, it was observed that the zebrafish showed significant wall-banging behavior 140 minutes after the start of the poisoning, which was an obvious escape behavior. The behavior lasted for about 1 hour; 220 minutes after the start of the poisoning, the zebrafish began to swim sideways intermittently; 230 minutes after the start of the poisoning, the zebrafish began to swim sideways continuously and the wall-banging behavior decreased. They swam in the activity area in a relatively stable and continuous state until the end of the experiment 5 hours after the start of the poisoning. The zebrafish were then fished out and placed in a pure water tank for recovery.

[0069] Embodiment 2:

[0070] like Figure 1 As shown, the present invention uses a self-developed pollutant exposure system, including a cylinder body with an open top, wherein the cylinder body is sequentially provided with three areas, namely, a buffer zone 1, a contaminated zone 2, and a water outlet zone 3. A water inlet partition 11 is provided between the buffer zone 1 and the contaminated zone 2, and a water outlet is provided between the contaminated zone 2 and the water outlet zone 3. The three areas are separated by partitions, and holes are punched on each partition to allow water and pollutants to pass through. The designed aperture is 2 mm. In order to enhance the pollutant diffusion effect, the distance between the holes is set to 1.5 times the aperture. All aquatic organisms smaller than this size can use the device for experiments. Each partition uses a fixed buckle made of acrylic sheet and a special suction cup for breeding (i.e., the first fixed suction cup 8) to fix the position of the partition. The buckle and suction cup should be placed on one side of the buffer zone 1 and the water outlet zone to prevent affecting the normal swimming and shooting of the organisms.

[0071] The buffer zone 1 is provided with a sewage inlet pipe 7, the other end of which is connected to a sewage tank that is filtered and kept at a constant temperature using a heating rod, and the outlet zone 3 is provided with an outlet pipe 13, the other end of which is connected to a wastewater collection system. The sewage inlet pipe 7 and the outlet pipe 13 are both connected to a peristaltic pump to provide power.

[0072] The buffer zone 1, the contaminated zone 2 and the water outlet zone 3 are provided with an internal circulation system.

[0073] The internal circulation system includes a submersible pump 10, a circulating water pipe 9 and an aeration assembly 4; the submersible pump 10 adopts the same submersible pump as the common freshwater fish farming filtration system on the market, and the circulation flow rate is 500-1500L / h, and the flow rate can be set within this range. The submersible pump 10 is placed in the middle of the cylinder wall in the water outlet area 3, and is fixed with an acrylic fixing buckle and a second fixing suction cup. The pump outlet is vertically upward, the water inlet section faces the cylinder wall, and maintains a distance of 3mm from the cylinder wall, and biochemical cotton is filled between the pump body and the cylinder body to absorb excrement.

[0074] The circulating water pipe is connected to the outlet of the submersible pump, and the other end is connected to the buffer 1, that is, the outlet of the circulating water pipe 9 is set in the buffer 1. A hole is opened 1 cm above the outlet of the circulating water pipe 9, and the aeration pipe 5 and the aeration regulating valve 6 are inserted, and the aeration regulating valve 6 is placed in the air. The interface uses a tubular hard plastic thin tube with a bevel cut, and the shorter side faces the outlet of the circulating water pipe 9 to ensure that the water flow does not enter the aeration pipe 5.

[0075] like Figure 2 As shown, as a further preference, the water inlet baffle 11 adopts a fully open-hole baffle, and the water outlet baffle 12 adopts a lower open-hole baffle.

[0076] The openings on each baffle can meet the conditions of water distribution systems and pollutants of different specifications. At the same time, this design can also meet the direct exposure of organisms that adapt to different water depths. For example, benthic mussels can use the lower perforated baffle.

[0077] A high-definition camera with a resolution of 1288×964 and a frame rate of 30 frames per second is selected, and the camera can clearly shoot the exposure system. The camera is installed and fixed using existing technology, and its installation position is located at a suitable height above the exposure system shown, so as to be able to shoot clearly.

[0078] In this embodiment, the design size of the cylinder body is 40×23×25cm, and the cylinder thickness is 5mm; each partition is vertically inserted into the cylinder body, dividing the long side of the cylinder into an 8cm buffer zone, a 22cm contaminated zone and a 9cm outlet zone; the sewage inlet pipe and outlet pipe are 12cm away from the bottom of the cylinder; the sewage inlet pipe is 1cm away from the outlet end of the circulating water pipe, and the aeration hole is 1cm away from the outlet end of the circulating water pipe; the bottom of the submersible pump is 8cm away from the bottom of the cylinder.

[0079] The partition size is 22×24cm, the bottom manure trough is 2mm high, the whole board is perforated with a hole diameter of 2mm and a thickness of 5mm.

[0080] The width of the swimming organisms used in the experiment is not less than 3mm, so the bottom feces trough and the aperture are both less than 3mm; the tank body is 23cm wide and the partition is 22cm wide. In the event of manufacturing errors, the fit is tight and can effectively prevent organisms with strong swimming ability from leaving the contaminated area from both sides of the partition; the partition height is designed to be slightly lower than the tank body height to prevent manufacturing errors from causing it to be higher than the tank body and affecting the subsequent installation of the cover; the cover is optional and its main purpose is to prevent fish from jumping out of the tank body.

[0081] Since fish and shrimps tend to jump out of the water, the water surface needs to be lower than the height of the baffle / tank to prevent them from leaving the contaminated area or jumping out of the device from above. To meet the vertical swimming distance of aquatic organisms, the water surface height is designed to be 20 cm, the size of the contaminated area is 22×23×20 cm, and the volume is 10.12 L.

[0082] The time-concentration variation function equation of the pollutant (chemical to be tested) in the three regions of the exposure system is as follows: Figure 3 As shown, the minimum effect concentrations corresponding to all abnormal behaviors were obtained through the concentration function of the contaminated area (the water directly exposed to the zebrafish) and the time when the abnormal behavior occurred.

[0083] like Figure 5 As shown, in this embodiment, the zebrafish 2D behavior analysis system of Viewpoint Company is used to collect the behavior information and data of zebrafish exposed to ZnSO4, and the specific method is as follows.

[0084] The 96h LC value of ZnSO4 on adult zebrafish was obtained by consulting the literature. 50 It is 461.54μM, and the mother liquor concentration is configured to be 450μM accordingly. This concentration of mother liquor is used as influent, and the influent flow rate is set to 1L / h.

[0085] The tank was pre-filled with pure water, which was preheated to 27°C using a heating rod, sterilized with an ultraviolet lamp and aerated for 1 hour; adult zebrafish (3 months) were placed in the contaminated area to adapt to the water quality, and the adaptation phase lasted for 1 hour; after the adaptation period, the peristaltic pump was turned on to connect pure water as the inlet water.

[0086] like Figure 6 The following is a diagram showing the trajectory of zebrafish at different times of ZnSO4 inflow. Turn on the Viewpoint behavioral analysis system camera and software, set the scale, set the minimum detection threshold to 20, the high-speed swimming threshold to 30, and the inactivity threshold to 5, collect data every 1 minute and 5 minutes, and start recording data after removing the background. First, conduct a 1-hour pure water inflow as a blank experiment, and its behavioral data is used as the baseline. After 1 hour, switch the water inlet pipe to the ZnSO4 mother solution to start the poisoning experiment. The experiment is carried out until the zebrafish can be seen to stop swimming almost to the naked eye, and the zebrafish is transferred to a pure water tank for recovery.

[0087] like Figure 6 It can be seen that in the blank control experiment within 1 hour, the swimming of zebrafish was concentrated in the center of the video acquisition area, and there was some wall-adhering swimming but it was less frequent. With the entry of pure water, there was no significant abnormal change in the behavior of zebrafish. When ZnSO4 began to enter the water, the trajectory range of zebrafish expanded significantly, and there were even cases where the trajectory left the acquisition area and entered a dead corner of the acquisition. This is a stress behavior, but overall the swimming speed and distribution of zebrafish did not change much. 2 hours after the start of the experiment, the movement trajectory changed significantly, the distance and number of high-speed swimming increased significantly, and abnormal patrolling states and other behavioral trajectories appeared. This phenomenon lasted for 1 minute, and then the zebrafish began to move in the central area of ​​the video acquisition area, with a slow swimming speed and a short swimming distance. This phenomenon lasted until the end of the experiment.

[0088] The moving distances of the three motion states, high-speed swimming, low-speed movement and near-stagnation state, are statistically analyzed as follows: Figure 7 As shown in the figure, as ZnSO4 began to enter the water, the overall swimming distance of the zebrafish gradually decreased at a lower speed, mainly reflected in the decrease in the low-speed moving distance. At 120 minutes, the high-speed moving distance and the low-speed moving distance increased significantly, and the moving distance in the near-stagnant state decreased significantly, indicating that the zebrafish had extremely significant abnormal behaviors under external stimuli, but because the behavior lasted only 1 minute and had no significant continuity before and after, this phenomenon does not rule out the interference of unknown external factors or individual differences in fish. Subsequently, the overall swimming distance of the zebrafish decreased to 1 / 2 of the baseline level after a slow decline of about 7 minutes, but it is worth noting that this decline is almost only reflected in the low-speed swimming distance, and the relative near-stagnant state movement distance increased significantly, which is consistent with the results of the experiment. Figure 6 The results of the images with the last time between 135-140min and 155-160min are consistent.

[0089] Combining the zebrafish's trajectory and the statistical data of zebrafish's behavior, we believe that zebrafish showed significant and continuous abnormal behavior of decreased activity 127 minutes after the start of the experiment, that is, 67 minutes after ZnSO4 began to enter the water. At this time, the concentration of ZnSO4 was 27.26% of the mother solution, that is, 122.67μM. This concentration is much lower than the 96h LC of ZnSO4 on zebrafish. 50 At this concentration, the zebrafish had no significant damage, but it can be concluded that this concentration of ZnSO4 has an inhibitory effect on the movement behavior of zebrafish. This concentration is far below the LC 50 , and it only took 127 minutes to produce a significant effect, which proved that this method can save experimental time and is also a non-destructive method to obtain toxicity data.

[0090] Example 3

[0091] like Figure 1 As shown, the present invention uses a self-developed pollutant exposure system, including a cylinder body with an open top, wherein the cylinder body is sequentially provided with three areas, namely, a buffer zone 1, a contaminated zone 2, and a water outlet zone 3. A water inlet partition 11 is provided between the buffer zone 1 and the contaminated zone 2, and a water outlet is provided between the contaminated zone 2 and the water outlet zone 3. The three areas are separated by partitions, and holes are punched on each partition to allow water and pollutants to pass through. The designed aperture is 2 mm. In order to enhance the pollutant diffusion effect, the distance between the holes is set to 1.5 times the aperture. All aquatic organisms smaller than this size can use the device for experiments. Each partition uses a fixed buckle made of acrylic sheet and a special suction cup for breeding (i.e., the first fixed suction cup 8) to fix the position of the partition. The buckle and suction cup should be placed on one side of the buffer zone 1 and the water outlet zone to prevent affecting the normal swimming and shooting of the organisms.

[0092] The buffer zone 1 is provided with a sewage inlet pipe 7, the other end of which is connected to a sewage tank that is filtered and kept at a constant temperature using a heating rod, and the outlet zone 3 is provided with an outlet pipe 13, the other end of which is connected to a wastewater collection system. The sewage inlet pipe 7 and the outlet pipe 13 are both connected to a peristaltic pump to provide power.

[0093] The buffer zone 1, the contaminated zone 2 and the water outlet zone 3 are provided with an internal circulation system.

[0094] The internal circulation system includes a submersible pump 10, a circulating water pipe 9 and an aeration assembly 4; the submersible pump 10 adopts the same submersible pump as the common freshwater fish farming filtration system on the market, and the circulation flow rate is 500-1500L / h, and the flow rate can be set within this range. The submersible pump 10 is placed in the middle of the cylinder wall in the water outlet area 3, and is fixed with an acrylic fixing buckle and a second fixing suction cup. The pump outlet is vertically upward, the water inlet section faces the cylinder wall, and maintains a distance of 3mm from the cylinder wall, and biochemical cotton is filled between the pump body and the cylinder body to absorb excrement.

[0095] The circulating water pipe is connected to the outlet of the submersible pump, and the other end is connected to the buffer 1, that is, the outlet of the circulating water pipe 9 is set in the buffer 1. A hole is opened 1 cm above the outlet of the circulating water pipe 9, and the aeration pipe 5 and the aeration regulating valve 6 are inserted, and the aeration regulating valve 6 is placed in the air. The interface uses a tubular hard plastic thin tube with a bevel cut, and the shorter side faces the outlet of the circulating water pipe 9 to ensure that the water flow does not enter the aeration pipe 5.

[0096] like Figure 2 As shown, as a further preference, the water inlet baffle 11 adopts a fully open-hole baffle, and the water outlet baffle 12 adopts a lower open-hole baffle.

[0097] The openings on each baffle can meet the conditions of water distribution systems and pollutants of different specifications. At the same time, this design can also meet the direct exposure of organisms that adapt to different water depths. For example, benthic mussels can use the lower perforated baffle.

[0098] A high-definition camera with a resolution of 1288×964 and a frame rate of 30 frames per second is selected, and the camera can clearly shoot the exposure system. The camera is installed and fixed using existing technology, and its installation position is located at a suitable height above the exposure system shown, so as to be able to shoot clearly.

[0099] In this embodiment, the design size of the cylinder body is 40×23×25cm, and the cylinder thickness is 5mm; each partition is vertically inserted into the cylinder body, dividing the long side of the cylinder into an 8cm buffer zone, a 22cm contaminated zone and a 9cm outlet zone; the sewage inlet pipe and outlet pipe are 12cm away from the bottom of the cylinder; the sewage inlet pipe is 1cm away from the outlet end of the circulating water pipe, and the aeration hole is 1cm away from the outlet end of the circulating water pipe; the bottom of the submersible pump is 8cm away from the bottom of the cylinder.

[0100] The partition size is 22×24cm, the bottom manure trough is 2mm high, the whole board is perforated with a hole diameter of 2mm and a thickness of 5mm.

[0101] The width of the swimming organisms used in the experiment is not less than 3mm, so the bottom feces trough and the aperture are both less than 3mm; the tank body is 23cm wide and the partition is 22cm wide. In the event of manufacturing errors, the fit is tight and can effectively prevent organisms with strong swimming ability from leaving the contaminated area from both sides of the partition; the partition height is designed to be slightly lower than the tank body height to prevent manufacturing errors from causing it to be higher than the tank body and affecting the subsequent installation of the cover; the cover is optional and its main purpose is to prevent fish from jumping out of the tank body.

[0102] Since fish and shrimps tend to jump out of the water, the water surface needs to be lower than the height of the baffle / tank to prevent them from leaving the contaminated area or jumping out of the device from above. To meet the vertical swimming distance of aquatic organisms, the water surface height is designed to be 20 cm, the size of the contaminated area is 22×23×20 cm, and the volume is 10.12 L.

[0103] The time-concentration variation function equation of the pollutant (chemical to be tested) in the three regions of the exposure system is as follows: Figure 3 As shown, the minimum effect concentrations corresponding to all abnormal behaviors were obtained through the concentration function of the contaminated area (the water directly exposed to the zebrafish) and the time when the abnormal behavior occurred.

[0104] In this example, potassium dichromate (K2Cr2O7) was used as a test chemical to determine its behavioral toxicity to zebrafish.

[0105] In this embodiment, the circulation flow rate is 1000L / h, and a heating rod is set in the buffer zone to maintain the stable temperature of the water in the cylinder, and the temperature is set to 25°C. At the same time, in order to enhance the anti-interference ability of the method, a soundproof box is used to hold the experimental cylinder, infrared rays are emitted through the infrared emission backplane, and an infrared camera is used to capture the video signal. Since the cylinder body is made of glass, the background in the video signal is grayish white (depending on the infrared intensity), and the zebrafish body does not penetrate infrared rays, so it appears black in the video signal. The trajectory is characterized and quantitatively output by identifying the change in the center of mass of black pixels.

[0106] The water flow rate was set to 1 L / h, i.e. 16.7 mL / min. The video was recorded as soon as the zebrafish was placed in the poisoned area. Pure water was introduced for 1 h. K2Cr2O7 was added at the 60th minute. The initial concentration of K2Cr2O7 was based on the median lethal concentration (LC50) of zebrafish at 96 h in the literature. 50 ) value (133.5 mg / L), the LC indicated in the literature 50 Cr 6+ The concentration of K2Cr2O7 was converted to the corresponding concentration for solution preparation. The K2Cr2O7 solution was added to the water for 4 hours. After the experiment, the zebrafish were placed in sterile aerated pure water for a month of recovery before the experiment could be carried out again.

[0107] In this experiment, no visually visible abnormal behavior is produced, and the reason is that the zebrafish wall-adhering movement is very frequent under the initial pure water state, which is related to the exploratory behavior of zebrafish after entering the new environment, and this behavior is affected by the individual's ability to adapt to the environment. Limited by the hardware limitation of video technology, there is deformation in the video image, and the side close to the camera is larger than the side away from the camera (the image gradually shrinks as the camera moves away radially), and the commercial video recognition software currently existing is mostly based on pixel point mass center change or machine learning to automatically identify fish feature parts, and then realize track tracking. Especially for the reflection produced by the transparent cylinder wall, the problems such as label abnormality, mass center drift may be produced, and further improvement is still needed. In the present embodiment, by the method of overall comparison and naked eye observation, the abnormal behavior produced and the corresponding minimum effect concentration are comprehensively determined, and the abnormal behavior of this type of special individual zebrafish is determined to be more suitable.

[0108] The distinguishing thresholds for each swimming state (high-speed swimming, low-speed swimming, and inactive swimming) are 30 cm and 5 cm, respectively. This method is used to count the time of each swimming state. Each statistical duration is 1 minute, and a total of 5 hours of behavioral analysis is performed. Video recording is achieved through an infrared camera. This embodiment uses Viewpoint's 2D ZebraTower software for video analysis. The junctions (adhesions and splicing) of the cylinder walls will appear black, which is close to the zebrafish. This results in the junctions of the cylinder walls being identified as "living things" when the entire water area is framed for analysis. This causes the center of mass identified by the zebrafish to drift and the trajectory to become chaotic. According to the usage and suggestions provided by the company, only the bottom plane of the cylinder is framed, which results in blind monitoring angles for the portion of each vertical surface close to the cylinder wall. Under normal monitoring conditions, the sum of the time for each swimming state per minute should be 60 seconds, while Figure 8 The parts less than 60s shown are all monitoring blind spots near the cylinder wall where the zebrafish swam, resulting in the loss of trajectory. Previous studies have shown that zebrafish will exhibit exploratory behaviors such as diving and swimming around the cylinder wall when entering a new environment; preliminary experimental results show that different zebrafish individuals have different exploratory behavior responses when entering the poisoning device described in this embodiment. Some fish swim normally in the cylinder after a short period of exploratory behavior, and the recorded movement trajectory is soft and relatively evenly distributed. At the same time, a small number of individuals have shown significant "escape" behavior since entering the poisoning device, that is, they keep swimming around the cylinder wall and partitions and try to swim outside the cylinder. This embodiment interprets the intensity of abnormal behavior of such a small number of individuals and calculates their minimum effect concentration.

[0109] The first 60 minutes were filled with pure water, which was intended to allow the zebrafish to adapt to the water quality and pump noise of the new environment and to record the normal behavior data of the zebrafish. Figure 8 As shown in the figure, the sum of the swimming state time in the first 60 minutes (recordable exercise time) is more than 30 seconds, and only a few time points are less than 30 seconds. However, due to the large number of data points, it is impossible to intuitively compare the changes and obtain the negative impact of K2Cr2O7. Therefore, an overall comparison is adopted to integrate the data into 5-minute recordable time, and then compare the data. The results are shown in the figure. Fig. 9 As shown. Fig. 9 It can be seen that during the pure water inlet stage, the shortest recordable exercise time was 33.9s and 31.2s at 45-50 and 50-55 minutes, and the rest of the time periods were higher than 36s. After K2Cr2O7 began to inlet water, the average recordable time began to drop below 30s at 110-115 minutes, and recovered to 52.6s at 180-185 minutes. During this period, except for the average value of 30.9s at 120-125 minutes, the average time was lower than 30s to varying degrees. Fig. 9It can be seen that from 65 to 70 minutes to 110 to 115 minutes, although there were certain fluctuations, the overall recordable movement time showed a downward trend, indicating that during this period, under the influence of K2Cr2O7, the wall-adherent swimming behavior of the test zebrafish gradually and fluctuatedly enhanced.

[0110] Previous studies have divided the behavior of aquatic organisms affected by toxic substances into "adaptation stage, adjustment stage and toxicity stage". In this embodiment, 65 to 115 minutes can be regarded as the "adaptation stage", when K2Cr2O7 enters the water, causing the zebrafish to sense abnormal water quality. Trace amounts of K2Cr2O7 activate the behavioral regulation of the zebrafish, causing them to gradually change their behavior and adapt to the new water quality; 110 to 185 minutes can be regarded as the "adjustment stage", when K2Cr2O7 enters the water, causing the zebrafish to sense abnormal water quality. Figure 8 It can be seen that the concentration of K2Cr2O7 tends to be stable in this stage and enters Figure 3 The slow rising stage shown in the figure, during this stage, the zebrafish tested is subjected to external pressure, and the endocrine system is adjusted to adapt to the new relatively steady-state environment. The "toxic stage" mentioned in previous studies refers to the serious action inhibition of zebrafish, which is usually manifested as no swimming or extremely slow swimming. This stage was not entered in the present embodiment. It is worth noting that the behavioral data after 185 minutes do not have a similar pattern as described above, but the overall trend of the total recordable time "rising first and then falling" is presented, and it is less than 30s after 260 minutes, and lasts for about 30 minutes. This stage does not have the characteristics of a new steady state after poisoning.

[0111] Since the average swimming time recorded every 5 minutes during the period of 110 to 185 minutes was less than 30 seconds, the behavioral data was significantly different from the blank control period, and the "adaptation phase" of 65 to 115 minutes was experienced, it can be considered that the concentration at 110 minutes is the minimum effective concentration of K2Cr2O7. When the time-concentration linear function is applied, the K2Cr2O7 concentration at this time is 26.46% of the mother solution, that is, 35.32 mg / L. The total time of this method is only 5 hours, which is much lower than the 96h LC 50 The test time is shorter than 1 second, and the amount of organism used is only 1. If the number of parallel controls is increased, it is also less than 10, and the test organism remains alive and has normal activities after the recovery period, the purpose of this patent is achieved.

[0112] Example 4

[0113] like Figure 1As shown, the present invention uses a self-developed pollutant exposure system, including a cylinder body with an open top, wherein the cylinder body is sequentially provided with three areas, namely, a buffer zone 1, a contaminated zone 2, and a water outlet zone 3. A water inlet partition 11 is provided between the buffer zone 1 and the contaminated zone 2, and a water outlet is provided between the contaminated zone 2 and the water outlet zone 3. The three areas are separated by partitions, and holes are punched on each partition to allow water and pollutants to pass through. The designed aperture is 2 mm. In order to enhance the pollutant diffusion effect, the distance between the holes is set to 1.5 times the aperture. All aquatic organisms smaller than this size can use the device for experiments. Each partition uses a fixed buckle made of acrylic sheet and a special suction cup for breeding (i.e., the first fixed suction cup 8) to fix the position of the partition. The buckle and suction cup should be placed on one side of the buffer zone 1 and the water outlet zone to prevent affecting the normal swimming and shooting of the organisms.

[0114] The buffer zone 1 is provided with a sewage inlet pipe 7, the other end of which is connected to a sewage tank that is filtered and kept at a constant temperature using a heating rod, and the outlet zone 3 is provided with an outlet pipe 13, the other end of which is connected to a wastewater collection system. The sewage inlet pipe 7 and the outlet pipe 13 are both connected to a peristaltic pump to provide power.

[0115] The buffer zone 1, the contaminated zone 2 and the water outlet zone 3 are provided with an internal circulation system.

[0116] The internal circulation system includes a submersible pump 10, a circulating water pipe 9 and an aeration assembly 4; the submersible pump 10 adopts the same submersible pump as the common freshwater fish farming filtration system on the market, and the circulation flow rate is 500-1500L / h, and the flow rate can be set within this range. The submersible pump 10 is placed in the middle of the cylinder wall in the water outlet area 3, and is fixed with an acrylic fixing buckle and a second fixing suction cup. The pump outlet is vertically upward, the water inlet section faces the cylinder wall, and maintains a distance of 3mm from the cylinder wall, and biochemical cotton is filled between the pump body and the cylinder body to absorb excrement.

[0117] The circulating water pipe is connected to the outlet of the submersible pump, and the other end is connected to the buffer 1, that is, the outlet of the circulating water pipe 9 is set in the buffer 1. A hole is opened 1 cm above the outlet of the circulating water pipe 9, and the aeration pipe 5 and the aeration regulating valve 6 are inserted, and the aeration regulating valve 6 is placed in the air. The interface uses a tubular hard plastic thin tube with a bevel cut, and the shorter side faces the outlet of the circulating water pipe 9 to ensure that the water flow does not enter the aeration pipe 5.

[0118] like Figure 2 As shown, as a further preference, the water inlet baffle 11 adopts a fully open-hole baffle, and the water outlet baffle 12 adopts a lower open-hole baffle.

[0119] The openings on each baffle can meet the conditions of water distribution systems and pollutants of different specifications. At the same time, this design can also meet the direct exposure of organisms that adapt to different water depths. For example, benthic mussels can use the lower perforated baffle.

[0120] A high-definition camera with a resolution of 1288×964 and a frame rate of 30 frames per second is selected, and the camera can clearly shoot the exposure system. The camera is installed and fixed using existing technology, and its installation position is located at a suitable height above the exposure system shown, so as to be able to shoot clearly.

[0121] In this embodiment, the design size of the cylinder body is 40×23×25cm, and the cylinder thickness is 5mm; each partition is vertically inserted into the cylinder body, dividing the long side of the cylinder into an 8cm buffer zone, a 22cm contaminated zone and a 9cm outlet zone; the sewage inlet pipe and outlet pipe are 12cm away from the bottom of the cylinder; the sewage inlet pipe is 1cm away from the outlet end of the circulating water pipe, and the aeration hole is 1cm away from the outlet end of the circulating water pipe; the bottom of the submersible pump is 8cm away from the bottom of the cylinder.

[0122] The partition size is 22×24cm, the bottom manure trough is 2mm high, the whole board is perforated with a hole diameter of 2mm and a thickness of 5mm.

[0123] The width of the swimming organisms used in the experiment is not less than 3mm, so the bottom feces trough and the aperture are both less than 3mm; the tank body is 23cm wide and the partition is 22cm wide. In the event of manufacturing errors, the fit is tight and can effectively prevent organisms with strong swimming ability from leaving the contaminated area from both sides of the partition; the partition height is designed to be slightly lower than the tank body height to prevent manufacturing errors from causing it to be higher than the tank body and affecting the subsequent installation of the cover; the cover is optional and its main purpose is to prevent fish from jumping out of the tank body.

[0124] Since fish and shrimps tend to jump out of the water, the water surface needs to be lower than the height of the baffle / tank to prevent them from leaving the contaminated area or jumping out of the device from above. To meet the vertical swimming distance of aquatic organisms, the water surface height is designed to be 20 cm, the size of the contaminated area is 22×23×20 cm, and the volume is 10.12 L.

[0125] The time-concentration variation function equation of the pollutant (chemical to be tested) in the three regions of the exposure system is as follows: Figure 3 As shown, the minimum effect concentrations corresponding to all abnormal behaviors were obtained through the concentration function of the contaminated area (the water directly exposed to the zebrafish) and the time when the abnormal behavior occurred.

[0126] Lead acetate is a chemical with extremely high aquatic toxicity. 2+ 24h LC of adult zebrafish 50 The initial concentration was 171 mg / L, and the initial concentration was appropriately increased to 200 mg / L (1.17 LC 50 ), and prepare lead acetate mother solution (313.98 mg / L) based on this concentration.

[0127] Fig.10The effect of lead acetate in water on the movement frequency, time and distance of zebrafish was shown. It can be found that Pb 2+ The number of movements, time and distance of each stage were affected to varying degrees. For the number of movements, the first 60 minutes of pure water inlet did not show a significant pattern, but the overall level was high, especially the number of inactive swimming was at a relatively high level (77.3±15.6 times) except for the 20th, 21st and 22nd minutes. At the 93rd minute after lead acetate was introduced into the water, the total number of movements decreased significantly, with 5 inactive swimming times and 5 low-speed swimming times, and no high-speed swimming times; at the same time, the swimming time diagram of each stage showed that within 60 seconds, the time in the inactive swimming state was 58.1 seconds, accounting for 96.8% of the analysis time, while before the beginning of lead acetate inlet, the inactive swimming state only accounted for (40.45±9.47)% of the analysis time; the same phenomenon appeared in the swimming distance, the swimming distance at the 93rd minute was 68.2 cm, and the average swimming distance from the 60th minute to the 93rd minute was 390.4±65.1 cm. At the same time, higher activity levels, increased swimming distances, and high-speed swimming states appeared at the 91st and 92nd minutes, all of which indicated that the zebrafish exhibited abnormal behavior at this time point. However, due to the short duration (5 minutes in total, including 2 minutes of recovery to normal) and the fact that no abnormal data significantly different from the baseline appeared except at the 93rd minute, it is impossible to directly determine that this concentration is the minimum effective concentration that causes abnormal behavior. It is worth noting that starting from the 100th minute, there was a significant loss of recordable movement time (<60s), indicating that the zebrafish began to swim against the wall. Since the zebrafish's movements were generally within the analysis area before, there was no loss of analysis time. Therefore, the time loss at this time reflects, to a certain extent, the behavior of the zebrafish escaping from the area, which lasted until the 152nd minute.

[0128] From 100 to 152 minutes, except for the escape behavior, the movement distance generally showed a slow downward trend ( Fig.10Neutron graph), but the number of movements did not show significant abnormal fluctuations. The decrease in movement distance and escape behavior indicate that the behavior of the test zebrafish has been affected by lead acetate, and this period can be considered an "adaptation stage." Since 152 minutes, the number of movements, movement time, and movement distance have all changed dramatically. First, the number of movements dropped sharply. The total number of movements in the pure water inlet stage, 60-100 minutes, and 100-152 minutes were 158.3±38.1, 177.3±50.8, and 180.3±31.1 times, respectively, while the total number of movements in 152-187 minutes was 8.2±7.9 times, a decrease of more than 94.8%; the inactivity time accounted for 6. The proportion of 0s also surged from (36.5±12.5)%, (43.1±14.4)%, and (34.9±13.9)% in the three stages to (98.1±2.3)%, an increase of more than 127.6%; the total swimming distance decreased from 427.4±118.5cm, 374.9±87.5cm, and 367.4±56.4cm in the three stages to 93.5±19.5cm, a decrease of more than 74.6%. The above statistics show that starting from the 152nd minute, the tested zebrafish underwent drastic behavioral changes and entered the "adjustment stage", with the endocrine system and nervous system working together to cause low activity. Although the zebrafish's movement intensity was relatively weak, it cannot be considered that the zebrafish has entered the "toxic stage" because the behavioral intensity of the test zebrafish began to recover to a certain extent at 204 minutes, and at 213 minutes into the experiment, the zebrafish jumped out of the water and entered the water outlet area with relatively low concentration in order to escape the contaminated area. This shows that the test zebrafish did not enter a state of toxic paralysis. After the test zebrafish were placed in pure water for cultivation, it was found that their behavioral state returned to normal in a relatively short period of time (about 1 day), and no deaths occurred.

[0129] In summary, it can be considered that at the 152nd minute of the total duration, the zebrafish produced significant behavioral toxicity effects, which were also reflected in the number of movements, movement time, and movement distance, and the behavioral intensity changed significantly and consistently. Through the time-concentration linear function, it was found that the lead acetate concentration at this time accounted for 28.44% of the mother liquor concentration, which was 89.28 mg / L (lead acetate concentration), Pb 2+ The concentration is 56.87 mg / L.

[0130] The whole experiment took 4 hours, which was much shorter than the LC 50 The test time is 24 hours, and no adverse effects such as death are produced on the tested organisms, which proves that the present invention is a non-destructive toxicity test method for obtaining toxicity data.

[0131] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A chemical toxicity testing method based on a self-developed pollutant exposure system, using a self-developed pollutant exposure system, the system includes a cylinder with an open top, a buffer zone, a contaminated zone and a water outlet zone are sequentially arranged inside the cylinder, the buffer zone and the contaminated zone, and the contaminated zone and the water outlet zone are separated by a perforated partition, and zebrafish is used as a model organism in the exposure system, characterized in that: The specific steps of the test method are as follows: (1) Prepare the master solution and query the LC of the chemical to be tested 50 Value, according to the LC 50 The initial concentration of the stock solution is prepared by adjusting the value. If the chemical has no obvious toxicity to zebrafish behavior, the concentration can be further increased according to the results of the preliminary experiment. (2) Establish a time-concentration linear function for the chemical to be tested in the exposure area, which is: When t ≤ 40 minutes, C = 0.60871t + 0.36129; When 40 minutes < t ≤ 600 minutes, C = 0.04706t + 24.10669; where C is the concentration percentage of the chemical relative to the mother liquor, in %, and t is the inlet time of the mother liquor; (3) Conduct a significant behavior test in the exposure system, and calculate the concentration at which the chemical to be tested causes significant changes in the behavior of zebrafish, that is, the lowest effective concentration; (4) Analyze the behavior of zebrafish in the exposure system by coupling camera tracing technology to obtain the lowest effective concentration of the chemical to be tested; Among them, steps (3) and (4) are selective analysis steps.

2. A chemical toxicity testing method based on a self-developed pollutant exposure system according to claim 1, characterized in that: The concentration of the starting chemical stock solution is based on the LC 50 The value is adjusted, the adjustment range is 0.5LC 50 ~2LC 50 .

3. A chemical toxicity testing method based on a self-developed pollutant exposure system according to claim 1, characterized in that: The partition distance of the self-developed pollutant exposure system is not less than 20 cm. The partition is perforated, and an internal circulation system is set in the buffer area, exposure area and water outlet area; an inlet pipe is set in the buffer area, and an outlet pipe is set in the water outlet area. The inlet pipe and outlet pipe are both connected to a peristaltic pump to provide power, and the inlet and outlet water flow rates are set at 0.1 - 10 L / h.

4. A chemical toxicity testing method based on a self-developed pollutant exposure system according to claim 3, characterized in that: The internal circulation system includes a submersible pump, a circulation water pipe and an aeration component. The aeration component includes an aeration pipe and an aeration regulating valve; the circulation flow rate of the submersible pump is 500 - 1500 L / h. The submersible pump is placed in the middle of the water outlet area cylinder wall and kept 1 - 3 mm away from the cylinder wall. Biochemical cotton is filled between its pump body and the cylinder body to absorb excrement; the circulation water pipe is connected to the outlet of the submersible pump, and the other end is connected to the buffer area. The outlet end of the circulation water pipe is vertically downward, and the outlet is located in the middle of the buffer area. A hole is opened 0.5 - 1 cm above the outlet of the circulation water pipe, and the aeration pipe and the aeration regulating valve are inserted, and the aeration regulating valve is placed in the air. The interface uses a tubular hard plastic thin pipe with a bevel cut, and the shorter side faces the outlet of the circulation water pipe to ensure that water flow does not enter the aeration pipe.

5. The chemical toxicity testing method based on a self-developed pollutant exposure system according to claim 3 is characterized in that: The partition includes an upper perforated partition, a lower perforated partition, and a fully perforated partition. The partition is made of transparent acrylic sheet, and a fecal tank with a height of 1 - 3 mm is left at the bottom.

6. A chemical toxicity testing method based on a self-developed pollutant exposure system according to claim 5, characterized in that: The aperture of the holes on the partition is 1 - 3 mm, and the distance between the holes is set to 1 - 2 times the aperture.

7. The chemical toxicity testing method based on a self-developed pollutant exposure system according to claim 1 is characterized in that: The time-concentration linear function of the chemical to be tested is obtained by fitting the scatter plot of the pollutant concentration and time measured at different time points in each area, and the fitting method is apparent fitting.

8. The chemical toxicity testing method based on a self-developed pollutant exposure system according to claim 1 is characterized in that: The method of the significant behavior test is as follows: Pre-fill pure water in the cylinder body, preheat it to 27°C with a heating rod, sterilize it with an ultraviolet lamp and aerate it for more than 1 h; place adult male zebrafish in the exposure area for water quality adaptation, and the adaptation stage is at least 1 h; after the adaptation period ends, conduct a blank control experiment for 1 h, that is, turn on the inlet and outlet peristaltic pumps, and the inlet peristaltic pump is connected to pure water. Observe the behavior of zebrafish during the blank control experiment in detail as the standard behavior control. If it is necessary to observe the toxicity target directionally, a corresponding toxicity target stimulation experiment should be additionally conducted; After the blank control experiment, the water inlet pipe is directly connected to the mother solution of the chemical to be tested to carry out the poisoning experiment. During the experiment, the normal behavior and abnormal behavior changes of the zebrafish are observed. The obvious abnormal postures need to be photographed and recorded, and the time when the abnormal behavior occurs is also recorded; If there is no significant abnormal behavior within 9 hours, increase the concentration of the test chemical stock solution and conduct a secondary experiment. If significant abnormal behavior occurs within 9 hours, record the time of the abnormal behavior and calculate the minimum effect concentration based on the time-concentration linear function of the test chemical.

9. A chemical toxicity testing method based on a self-developed pollutant exposure system according to claim 8, characterized in that: The standard / normal behaviors include sinking, floating, cruising, and hitting the wall; the abnormal behaviors include sudden high-speed movement, long-term abnormal high-speed / static state, and changes in swimming posture.

10. A chemical toxicity testing method based on a self-developed pollutant exposure system according to claim 1, characterized in that: For the test chemicals with lower toxicity, their behavioral changes are analyzed through coupled video tracing technology to obtain non-lethal abnormal behavior concentration, that is, the minimum effect concentration.

11. A chemical toxicity testing method based on a self-developed pollutant exposure system according to claim 10, characterized in that: The behavior analysis method of the coupled video-tracing technique is: Install a camera at a suitable height above the center of the cylinder to take pictures of the inside of the cylinder; First, shoot a 1-2 hour blank control video as a normal baseline, and then add the chemical mother solution into the water until the zebrafish behavior becomes abnormal or the chemical mother solution has been added to the water for 10 hours; The zebrafish was fished out and placed in sterilized pure water to recover. The video was analyzed to quantitatively obtain data on the swimming trajectory, swimming speed, distance and time of the zebrafish. By comparing with the baseline, long-term abnormal movement state was selected as an indicator of abnormal behavior, and the minimum effective concentration was calculated based on the starting time of this abnormal movement state.

12. A chemical toxicity testing method based on a self-developed pollutant exposure system according to claim 11, characterized in that: A high-definition camera with a resolution of at least 1288×964 and a frame rate of 30 frames per second is selected, and the camera can clearly capture the exposed system.

Images